Challenge of Pigs with Classical Swine Fever Viruses after C-Strain Vaccination Reveals Remarkably Rapid Protection and Insights into Early Immunity

Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser® C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-γ responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-γ response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination

Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses.

A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping

Primer and probes used in the triplex RT-PCR.

<p>Primer and probes used in the triplex RT-PCR.</p

Viruses used to determine analytical specificity of the triplex RT-PCR assay.

<p>Viruses used to determine analytical specificity of the triplex RT-PCR assay.</p

Comparison of triplex RT-PCR with reference RT-PCR and PCR assays for detection of CSFV and ASFV in experimental and field samples.

<p>Comparison of triplex RT-PCR with reference RT-PCR and PCR assays for detection of CSFV and ASFV in experimental and field samples.</p

Analytical sensitivity of triplex and reference method PCRs on samples from experimentally infected animals.

<p>(A) Viral RNA, extracted from blood samples taken at various time points post challenge from 2 animals (circles or squares) experimentally infected with CSFV and analysed by triplex RT-PCR (filled symbols) or CSFV RT-nPCR-TaqMan assay (open symbols). (B) Viral RNA, extracted from homogenised spleen samples taken from 6 pigs euthanized at various time points post challenge with ASFV and analysed by triplex RT-PCR (filled symbols) and ASFV-PCR (open symbols).</p

Diagnostic sensitivity and specificity estimates of triplex RT-PCR compared to reference RT-PCR and PCR assays for CSFV and ASFV.

<p>Diagnostic sensitivity and specificity estimates of triplex RT-PCR compared to reference RT-PCR and PCR assays for CSFV and ASFV.</p

Kinetics of vaccine induced prevention or reduction of viral RNA in blood and nasal secretions.

<p>Viral RNA concentrations, determined by real time RT-PCR, in blood (solid lines) and nasal swab samples (dashed lines). A) High levels of viral RNA were detected in blood soon after challenge in unvaccinated animals (open circles), with nasal secretions becoming RNA positive 5–6 days later. B) No viral RNA, or very low levels in the inconclusive range of the assay, was detected in blood or nasal secretion of animals vaccinated 5 days prior to challenge (filled circles). C) High levels of viral RNA was detected in blood and nasal swab samples of one of the animals vaccinated 3 days prior to challenge with UK2000/7.1. An intermediate level of viral RNA was present in blood of one animal vaccinated 3 days prior to challenge with CBR/93 (open squares), whereas the remaining animals in these groups in both experiments (filled squares) had either a transient low level, or no, viral RNA in blood, and either no or intermediate levels of RNA in nasal secretions. D) Animals vaccinated one day prior to challenge either developed clinical disease and were euthanized by 18 dpc (open triangles) or remained healthy for the duration of the experiment (filled triangles).</p

Pathological parameters.

a)<p>Number of animals that developed clinical signs and were euthanized early or had no or few signs and survived until the end of the experiment.</p><p>*Significant difference to unvaccinated control group (P<0.05).</p

Vaccine induced protection against leukopenia.

<p>Leucokyte numbers remained at pre-vaccination levels throughout the experiment in animals vaccinated five days prior to challenge with A) UK2000/7.1 or B) CBR/93 (filled circles), whereas a significant (P<0.05) decrease occurred rapidly after challenge in unvaccinated animals (open circles) and animals vaccinated one day before challenge that developed clinical signs and were euthanized prior to the end of the experiment (open triangles). A smaller, non significant, decrease in leukocyte numbers was observed in animals vaccinated one day prior to challenge that did not develop clinical signs (filled triangles) and those vaccinated three days prior to challenge (filled squares). n = number of animals in groups vaccinated on day prior to challenge that either developed signs and were euthanized or remained clinically healthy. Data are mean leukocyte counts for each group of pigs. Error bars represent SEM.</p